1. Field
The invention is in the field of automated equipment for staining specimens on glass slides.
2. State of the Art
There are currently many medical diagnostic tests performed by applying a biological material, such as blood, pus, urine or a bacterial culture, on a glass slide, such as a microscope slide, by smearing the material onto the slide, and then evaluating the material applied to the slide. In most instances, the smear will be treated with some type of reagent, such as a stain, to bring out or make visible various features of the smear not otherwise visible. In most cases the smear will also be fixed to the slide prior to or concurrently with staining.
It is currently normal practice with hematology smears, i.e. blood smears, to place a smear on a slide and allow the smear to dry. The smear is then fixed on the slide by wetting the smear with anhydrous methanol and allowing the methanol to evaporate or dry. Dye is then applied to the smear to bring out desired characteristics of the smear and the smear is then examined. While the staining intensity achieved depends on the time and the amount of stain applied, considerable variation in those variables still yield satisfactory results.
In addition to blood smear staining, it is common to observe bacteria in specimens and products as an indication of infection or contamination. Material to be observed is usually applied to a microscope slide, usually by smearing the material on the slide, fixed, and stained using the well known gram staining procedure. The gram staining consists of saturation of the smear with crystal violet followed by a thorough water wash, and then saturation with iodine followed by a thorough water wash. These steps are non-critical and as long as enough crystal violet and iodine are used to saturate the smear, any extra amount used does not affect the results obtained. The critical step in gram staining is decolorization. This is done by applying an organic solvent to selectively remove the crystal violet-iodine complex from the gram negative, but not the gram positive organisms. A counterstain containing safranin is then applied to stain the gram negative organisms red.
The rate of removal of the complex is species specific. If too little decolorizer is added the gram negative organisms (red) will appear gram positive (blue). If too much is added, the gram positive organisms will appear gram negative. Reliable decolorization can only be achieved where the amount of solvent applied to the smear is accurately controlled.
U.S. Pat. Nos. 4,004,550 and 4,089,989 show an automated slide stainer which automatically applies stain to slides mounted on a carousel for rotation in a staining chamber. A commercial version of the stainer shown in such patents is manufactured by Wescor, Inc., Logan, Utah and sold under the trademark Aerospray.
In the commercial version of the stainer shown in the above cited patents, slides to be stained are placed in a slide holding carousel which is rotated by a motor, as shown in the patent. During rotation of the carousel, the slides are sprayed with various reagents as they move past a spray nozzle, again as shown in the patents. However, it is impractical to use aerosol cans, as shown in the patents, for spraying the various reagents. Rather, pumps are provided for each of the reagents to pump the reagents from their container or other reservoir through a check valve to prevent back flow and through a nozzle which atomizes the reagent and sprays it onto the slides in the spray chamber. In order to spray a reagent onto the slide, the pump for the appropriate reagent is energized. The reagent is then sprayed into the chamber and onto the slides during the time its pump is energized. The pumps are timed and controlled to pump the proper reagents into the spray chamber at the proper times and for the proper length of time. With such system, however, the spray continues to flow into the chamber for 1 to 1.5 seconds during wind-down of the pump and depressurization of the delivery line after power to the pump is cutoff. This prevents highly accurate application of a reagent to the slides. If the slide carousel rotates at 30 RPM, an additional one second of spray application during wind down of the pump will give half of the slides in the carousel an extra application of reagent. Furthermore, the application of reagent to the slides is not uniform as the pump looses pressure. This means that some of the slides will receive an extra application of reagent and some of the slides will receive uneven dribble. In the case of hematology staining, as indicated above, this unevenness does not seem to be a significant problem. The staining approaches a saturation point and differences in staining due to this uneven application are not detectable. However, in gram staining, the application of the decolorization solvent must be accurately controlled and the extra application of such solvent to some of the slides and not others affects the accuracy and reproducibility of the gram staining results. Thus, the currently commercially available automated staining equipment made according to the referenced patents cannot be used for optimal gram staining.
In addition to the problem encountered if such equipment is used for gram staining, the additional stain used during wind down of the pump wastes the various reagents used. Further, because of the wind down time, it has not been practical to try to program the equipment for use with less than a full load of slides. Thus, the equipment is set to provide full pressure spray for the appropriate number of full revolutions of the carousel, regardless of how many slides are actually in the carousel. If the carousel is built to hold twelve slides, and only one or two slides are to be treated, the equipment will still spray enough reagent into the spray chamber to treat twelve slides, thus additionally wasting reagent.